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Syndecan-4 promotes cytokinesis in a phosphorylation-dependent manner

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Abstract

During mitosis, cells detach, and the cell–matrix interactions become restricted. At the completion of cytokinesis, the two daughter cells are still connected transiently by an intercellular bridge (ICB), which is subjected to abscission, as the terminal step of cytokinesis. Cell adhesion to the matrix is mediated by syndecan-4 (SDC4) transmembrane heparan sulfate proteoglycan. Our present work demonstrated that SDC4 promotes cytokinesis in a phosphorylation-dependent manner in MCF-7 breast adenocarcinoma cells. The serine179-phosphorylation and the ectodomain shedding of SDC4 changed periodically in a cell cycle-dependent way reaching the maximum at G2/M phases. On the contrary, the phospho-resistant Ser179Ala mutant abrogated the shedding. The phosphorylated full-length and shed remnants enriched along the mitotic spindles, and subsequently in the ICBs, however, proper membrane insertion was necessary for midbody localization. Expression of phosphomimicking Ser179Glu SDC4 resulted in incomplete abscission, whereas expression of the phospho-resistant SDC4 led to giant, multinucleated cells.

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Acknowledgments

The authors are indebted to Dr. Ibolya Kiss to share the cell culture facilities and Edit Kotogany for the technical assistance in the FACS experiments. A.K.-P. received a fellowship from the Gedeon Richter Centenary Foundation. This work was supported by grants KFP1-00024/2005 to L.S; NKTH-OM-00087/2007 and OTKA T049608 to F.D.; OTKA NK72595 to J.T.

Author information

Correspondence to Laszlo Szilak.

Electronic supplementary material

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Supplementary Video 2. Time-lapse, phase-contrast video of asynchronous proliferating MCF-7 cells expressing Ser179Ala. The proliferating cells were rounded up similarly to the non-transfected MCF-7 cells. Time-lapse photos were taken every 5 min at 25°C; frame rate was 30 frames per second (fps). (MPG 2412 kb)

Supplementary Video 3. Time-lapse, phase-contrast video of asynchronous proliferating MCF-7 cells expressing Ser179Glu mutant syndecan-4. The proliferating cell is rounded up but remained connected to the neighboring cells by permanent cytoplasmic bridges seen as thin thread. Time-lapse photos were taken every 5 min at 25°C; frame rate was 30 frames per second (fps). (MPG 720 kb)

Supplementary Video 4. Time-lapse, phase-contrast video of asynchronous proliferating MCF-7 cells expressing DEGE construct. The proliferating cells were rounded up but remained connected to the neighboring cells by permanent cytoplasmic bridges. Time-lapse photos were taken every 5 min at 25°C; frame rate was 30 frames per second (fps). (MPG 1218 kb)

Supplementary Figure 1. Control experiments. (a) Negative controls for immunofluorescence stainings. To corroborate that the secondary antibodies reacted specifically with the primary antibodies the samples were incubated with Fab fragments of anti-goat antibody conjugated with Cy2 and anti-rabbit antibody conjugated with Cy3 raised in donkey. Nuclear staining is blue. (b) Establishment of the specificity of the anti-SDC4-endo antibody. The lysates of wt-SDC4-GFP expressing cells were subjected to SDS-PAGE and blots were developed with anti-GFP (Zymed) and anti-SDC4-endo antibodies, respectively. Both antibodies similarly highlighted the full-length, GFP-tagged wt-SDC4-GFP monomers. However, the anti-SDC4-endo antibody visualized the 13 kDa band representing the shed remnant. (TIFF 235 kb)

Supplementary Figure 2. The proliferation rate of syndecan-4 transfected cell lines. Cell proliferation was measured in cultures of non-transfected, wt-SDC4-GFP, Ser179Ala and Ser179Glu expressing MCF-7 cells using the CellTiter96 AQueous One solution cell proliferation assay according to instruction of the supplier (Promega, Madison, WI). Briefly, 1,500 cells per well were plated in 96-well plates and cultured in 180 µl medium. After 16 h of culturing, the medium was changed and 20 µl reagent containing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was added. The amount of formazan product, indicative of the mitochondrial function of living cells and cell viability, was measured at 492 nm (TIFF 103 kb)

Supplementary Video 1. Time-lapse, phase-contrast video of asynchronous proliferating non-transfected MCF-7 cells. During the mitosis the proliferating cells were rounded up and separated from the neighboring cells. Time-lapse photos were taken every 5 min at 25°C; frame rate was 30 frames per second (fps). (MPG 1484 kb)

Supplementary Video 2. Time-lapse, phase-contrast video of asynchronous proliferating MCF-7 cells expressing Ser179Ala. The proliferating cells were rounded up similarly to the non-transfected MCF-7 cells. Time-lapse photos were taken every 5 min at 25°C; frame rate was 30 frames per second (fps). (MPG 2412 kb)

Supplementary Video 3. Time-lapse, phase-contrast video of asynchronous proliferating MCF-7 cells expressing Ser179Glu mutant syndecan-4. The proliferating cell is rounded up but remained connected to the neighboring cells by permanent cytoplasmic bridges seen as thin thread. Time-lapse photos were taken every 5 min at 25°C; frame rate was 30 frames per second (fps). (MPG 720 kb)

Supplementary Video 4. Time-lapse, phase-contrast video of asynchronous proliferating MCF-7 cells expressing DEGE construct. The proliferating cells were rounded up but remained connected to the neighboring cells by permanent cytoplasmic bridges. Time-lapse photos were taken every 5 min at 25°C; frame rate was 30 frames per second (fps). (MPG 1218 kb)

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Keller-Pinter, A., Bottka, S., Timar, J. et al. Syndecan-4 promotes cytokinesis in a phosphorylation-dependent manner. Cell. Mol. Life Sci. 67, 1881–1894 (2010). https://doi.org/10.1007/s00018-010-0298-6

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Keywords

  • Syndecan-4
  • Phosphorylation
  • Shedding
  • Cytokinesis
  • Intercellular bridge
  • Midbody
  • Multinucleation